-
[show abstract]
[hide abstract]
ABSTRACT: To test whether the functional impairment of the host bone marrow (BM) microenvironment pre-existing at the time of transplantation could be overcome by the increased content of immature cells in allogeneic peripheral blood stem cell transplantation (PBSCT) when compared with bone marrow transplantation (BMT).
Cobble stone area forming cells (CAFC) were assayed in normal BM and BM after allogeneic BMT and PBSCT after stable engraftment. Groups were compared by two-tailed t-test.
While BM from 11 normal controls contained an average of 778.8 CAFC-d35 per 10(6) low density bone marrow cells (LDBMC, range 453-1231 per 10(6) LDBMC), BM from patients after BMT contained an average of 123.7 CAFC-d35 per 10(6) LDBMC (range 38-257) per 10(6) LDBMC. BM from patients transplanted with PBSC after myeloablative conditioning contained 128.3 (range 46-305) CAFC-d35 per 10(6) LDBMC (P = 0.89 compared with BMT). Similar results were obtained when patients after PBSCT with non-myeloablative conditioning were included (P = 0.62 compared with BMT). CAFC numbers in patients transplanted in early stages of myeloid leukaemia (acute myeloid leukaemia first remission, chronic myeloid leukaemia first chronic phase) were significantly higher than CAFC numbers in patients transplanted in more advanced stages (P = 0.008) or myelodysplastic syndrome (P = 0.023). The lowest CAFC numbers were found in two cases of retransplantation.
Our findings indicate that the functional state of the BM microenvironment rather than stem cell dose or source is limiting for the homing and engraftment of immature haemopoietic cells in clinical transplantation.
European Journal Of Haematology 08/2004; 73(1):1-9. · 2.61 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The existence of functional gap junctions between haematopoietic progenitor cells (HPCs) and stromal cells of the haematopoietic microenvironment in the human system is a controversial issue. Primary CD34+ HPCs isolated from leukapheresis products were co-incubated with the human fibroblastoid bone marrow stromal cell line L87/4 in short-term liquid culture. Using the highly sensitive double whole-cell patch-clamp technique, we found that the majority (91%) of CD34+ HPCs are electrically coupled to L87/4 cells. Importantly, efficient coupling was observed within 1 h of the attachment of CD34+ HPCs to plastic adherent L87/4 cells. By comparison, homologous cell pairs formed by L87/4 cells exhibited a significantly higher electric coupling. Analysis of single-channel conductances revealed an electric profile characteristic of connexin 43 (Cx43)-type gap junctions for both homologous and heterologous cell pairs. The Cx phenotype was confirmed using Cx43-specific monoclonal antibodies in a flow cytometric assay and reverse transcription polymerase chain reaction (RT-PCR) for the detection of Cx43 mRNA. Finally, the electrophysiological studies were complemented by dye-transfer experiments using the recently described 'parachute' technique that allows the monitoring of dye diffusion without disruption of the plasma membrane. Taken together, our data indicate that functional Cx43-type gap junctions exist between stromal cells and immature HPCs and, thus, may provide an important regulatory pathway in haematopoiesis.
British Journal of Haematology 12/2000; 111(2):416-25. · 4.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We compared the biological effects of the CXC chemokine SDF-1alpha on immunomagnetically purified CD34+ cells isolated from human normal bone marrow (NBM), leukapheresis products (LP) and patients with chronic myeloid leukaemia (CML). LP CD34+ cells showed a significantly stronger migration response to SDF-1alpha (100 ng/ml) than CD34+ cells isolated from the peripheral blood (PB) of CML patients (P < 0.05). The chemotactic response to SDF-1alpha was also reduced in CML BM CD34+ cells in comparison to NBM CD34+ cells but the observed differences were not statistically significant. In analogy to normal CD34+ cells circulating CML PB CD34+ cells were less responsive to SDF-1alpha than their BM counterparts (P < 0.05). Furthermore, SDF-1alpha elicited similar concentration-dependent growth suppressive effects on normal and CML CD34+ cells (P > 0.05) in colony-forming cell assays. We then demonstrated that SDF-1alpha triggers intracellular calcium increases in CD34+ cells and there were no differences in the time course and dose response characteristics of normal and CML CD34+ cells. The reduced migration response to SDF-1alpha in CML CD34+ cells was not due to a down-regulation of the SDF-1alpha receptor CXCR-4 as flow cytometric analysis revealed similar CXCR-4 expression levels on NBM, LP, CML PB and CML BM CD34+ cells (P > 0.05). Finally, no differences in the modulation of CXCR-4 levels in response to SDF-1alpha and serum were observed in CML and normal CD34+ cells. Our data suggest that the impaired chemotactic response of CML CD34+ cells to SDF-1alpha is not caused by a lack or complete uncoupling of CXCR-4, but may be due to an intracellular signalling defect downstream of the receptor.
Leukemia 09/2000; 14(9):1652-60. · 9.56 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The cellular isoform of the prion protein (PrPC) is a cell surface glycoprotein that has recently been shown to play a role in haemopoietic cell activation and proliferation. We have characterized the constitutive expression of PrPC on human peripheral blood (pB) cell populations, using PrP-specific antibodies in a multiparameter flow cytometry approach. We found that T cells, NK cells and monocytes exhibit similar PrPC levels, whereas PrPC surface staining on B cells was significantly lower and was virtually absent on granulocytes. Within the T-cell compartment, CD8+ cells showed a significantly higher PrPC expression than CD4+ cells. Similarly, CD3+ cells co-expressing the activation marker CD56 (N-CAM) exhibited significantly higher PrPC expression levels than their CD56- counterparts. Culture of CD14+ pB monocytes for 12-48 h in the presence of interferon gamma (IFN-gamma) resulted in a significant increase in PrPC expression in a time- and concentration-dependent manner. This effect was partially abrogated by the addition of the metabolic inhibitor cycloheximide, indicating the role of protein synthesis in this process. Our results show that PrPC expression on human haemopoietic cells correlates with the activation and developmental status of these cells, suggesting an important functional role of PrPC in the haemopoietic system.
British Journal of Haematology 04/2000; 108(3):488-95. · 4.94 Impact Factor
